Fortunellin ameliorates LPS‐induced acute lung injury, inflammation, and collagen deposition by restraining the TLR4/NF‐κB/NLRP3 pathway

Abstract Objective Acute lung injury (ALI) is the prevalent respiratory disease of acute inflammation with high morbidity and mortality. Fortunellin has anti‐inflammation property, but its role in ALI remains elusive. Thus, this study clarified the function of fortunellin on ALI pathogenesis. Methods The ALI mouse model was established by lipopolysaccharide (LPS) induction, and lung tissue damage was evaluated utilizing hematoxylin–eosin (HE) staining. The edema of lung tissue was measured by the lung wet/dry (W/D) ratio. The lung capillary permeability was reflected by the protein content in bronchoalveolar lavage fluid (BALF). Inflammatory cell infiltration was measured by the evaluation of the content of myeloperoxidase (MPO), neutrophils, and leukocytes in BALF. Cell apoptosis was measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The secretions of inflammatory cytokines were quantified using enzyme‐linked immunosorbent assay (ELISA) assays. Lung tissue collagen deposition was evaluated by Masson staining. Results Fortunellin attenuated LPS‐induced lung tissue damage and reduced the W/D ratio, the content of MPO in lung tissue, the total protein contents in BALF, and the neutrophils and leukocytes number. Besides, fortunellin alleviated LPS‐stimulated lung tissue apoptosis, inflammatory response, and collagen deposition. Furthermore, Fortunellin repressed the activity of the Toll‐like receptor 4 (TLR4)/nuclear factor kappa‐B (NF‐κB)/NLR Family Pyrin Domain Containing 3 (NLRP3) pathway in the LPS‐stimulated ALI model and LPS‐induced RAW264.7 cells. Moreover, fortunellin attenuated LPS‐stimulated tissue injury, apoptosis, inflammation, and collagen deposition of the lung via restraining the TLR4/NF‐κB/NLRP3 pathway. Conclusion Fortunellin attenuated LPS‐stimulated ALI through repressing the TLR4/NF‐κB/NLRP3 pathway. Fortunellin may be a valuable drug for ALI therapy.


| INTRODUCTION
Acute lung injury (ALI) is a severe respiratory disease with high morbidity and mortality annually. 13][4] ALI has a complex etiology and can be caused by sepsis, pneumonia, burns, chest trauma, and other factors. 5Despite a large number of clinical trials and research conducted, there is still no effective strategy for treating ALI due to the continuous increase in drug resistance and the emergence of new pathogens, such as the global pandemic of novel coronavirus pneumonia. 6Therefore, ALI remains a significant threat to public health.It is urgent to investigate new drugs for ALI therapy.
Fortunellin (acacetin 7-O-neohesperidoside, Figure 1A) is a citrus flavonoid extracted from the fruit of Fortunella margarita (kumquat). 7Kumquat extracts usually possess anticancer, antioxidant, and anti-inflammation properties. 8ccumulating evidence reveals that fortunellin has similar functions to kumquat extracts and is regarded as a valuable anti-inflammation agent. 7,9,10For example, Zhao et al. reported that fortunellin improved the histopathological alterations and heart function and suppressed inflammatory response in diabetic cardiomyopathy. 7Xiong et al. found that fortunellin restrained the dysregulated inflammation and inhibited cell apoptosis in colitis. 9Nevertheless, the function of fortunellin on ALI remained elusive.Based on the potential function of fortunellin in inflammation regulation, we inferred that fortunellin might regulate ALI.Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB)/NLR Family Pyrin Domain Containing 3 (NLRP3) pathway is critical in the regulation of inflammation and apoptosis. 11,12Bai et al. demonstrated that Biochanin A suppressed inflammation by restraining the TLR4/NF-kB/NLRP3 pathway during myocardial ischemia/reperfusion injury. 11Luo et al. proved that Ginsenoside Rg1 ameliorated cardiomyocyte inflammation by repressing the TLR4/NF-kB/NLRP3 pathway. 12esides, this pathway also participated in the ALI progression. 13,14For instance, glycoprotein improved lipopolysaccharide (LPS)-stimulated ALI via blocking TLR4/NF-κB/NLRP3 pathway. 13Avanafil attenuated LPS-stimulated ALI via downregulating the TLR4/NF-κB/NLRP3 pathway. 14Therefore, we speculated that fortunellin might prevent ALI progression by modulating the TLR4/NF-κB/NLRP3 pathway.
Hence, this study aimed to elucidate whether fortunellin could alleviate ALI progression, including tissue injury, apoptosis, inflammation, and collagen deposition of the lung, in an LPS-induced ALI model and explore if the TLR4/NF-κB/NLRP3 pathway mediated the regulation process.

| Animals and treatments
C57BL/6 mice aged 6-8 weeks were acquired from Beijing Vital River Laboratory Animal Technology Co., Ltd.The animal research complied with national and international regulations and policies.This study was authorized by The Ethics Committee of the Affiliated Hospital of Putian University (No. 202013).To generate adenovirus carrying expression vectors for TLR4 (ad-TLR4), TLR4 plasmid was blunt-ended and cloned into the shuttle vector pAd5/CMVk-NpA using the EcoRV site, which was completed at GenePharma.
Sixty mice were divided into five groups: sham, LPS, LPS + 10 mg/kg fortunellin (FOR), LPS + 20 mg/kg FOR, LPS + 30 mg/kg FOR groups.There are 12 mice in each group.The mice in the LPS group received intratracheal instillation of LPS (5 mg/kg) (Escherichia coli 055: B5, #L2880, Sigma). 15Briefly, mice were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium, and a midline incision was made in the neck to expose the trachea.After that, a 28-gauge needle was inserted into the trachea above the carina, and LPS was instilled.After intratracheal instillation, mice were placed vertically and rotated for 1 min to ensure even lung distribution.The mice in the sham group were given an equal volume of distilled phosphate-buffered saline (PBS).The mice in LPS + 10 mg/kg FOR, LPS + 20 mg/kg FOR, and LPS + 30 mg/kg FOR groups received 10, 20, and 30 mg/kg of fortunellin (Purity speciation: ≥98%) (BioCrick) via intragastric administration before 1 h of LPS administration (5 mg/kg), respectively.The left lung of six mice in each group was used for bronchoalveolar lavage fluid (BALF) collection, and the right lung was used for lung wet/dry (W/D) weight ratio determination.The right lung of the other six mice in each group was used for histological analysis using Hematoxylin-eosin (HE) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and Masson staining, and the left lung was used for myeloperoxidase (MPO) activity determination (left upper lobe) and western blot (left lower lobe).
The other 24 mice were allocated into sham, LPS, LPS + 30 mg/kg FOR, and LPS + 30 mg/kg FOR+ad-TLR4 groups.There are six mice in each group.The mice in LPS + 30 mg/kg FOR+ad-TLR4 groups were injected with ad-TLR4 (1×10 9 PFU/mouse) via the tail vein 7 days before other treatments.Mice were euthanasia utilizing CO 2 inhalation at 12 h after LPS inducement.The left lung of mice in each group was used for BALF collection, and the right lung was used for histological analysis using HE staining (right upper lobe) and western blot (right lower lobe).

| Histological analysis of lung tissue
Lung tissues were immobilized, embedded, and prepared into 5 µm slices.Then the sections were dyed utilizing hematoxylin for 5 min and eosin for 1 min.The results were evaluated using a light microscope (DM2500, Leica).The lung histopathological changes were scored using the scoring criteria as previously described. 16

| Lung W/D weight ratio
The right lung tissues of mice were harvested and weighed as wet weight.After that, issues were dried at 80°C for 48 h and weighed as dry weight.The wet/dry ratio was then determined to estimate the edema.

| MPO activity determination
Lung tissues were homogenized and centrifuged at 4°C.The supernatant was harvested to evaluate the MPO activity at 460 nm utilizing the MPO Detection Kit (Nanjing Jiancheng Bioengineering Institute) in compliance with the suppliers' instructions.The results were exhibited as units per gram of total protein (U/g).
BALF was harvested by lavaging the left lung using PBS and centrifugation.Afterward, the cell pellet and supernatant were gathered, respectively.The cell pellet was stained with Wright-Giemesa to test neutrophils, leukocytes, and monocytes, and the numbers of neutrophils, leukocytes, and monocytes were analyzed utilizing a microscope (DM2500, Leica).The supernatant was applied to protein content analysis utilizing the bicinchoninic acid (BCA) method.

| Lung function test
Lung function injury indexes, including lung injury score, minute ventilation (mL/kg), lung volume (mL), and airway resistance, were estimated according to the previous report. 17

| Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining
The lung tissues were immobilized, embedded, and prepared into 5 µm slices.The slices were deparaffinized, rehydrated, and blocked endogenous peroxidase activity.After that, the sections were used to assess lung tissue apoptosis utilizing the Colorimetric TUNEL Apoptosis Assay Kit (Beyotime) in compliance with the supplier's protocols.The apoptosis was measured under a microscope (DM2500, Leica).
2.9 | Enzyme-linked immunosorbent assay (ELISA) assays BALF was harvested by lavaging the left lung using PBS and centrifugation at 300 g.The productions of IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in BALF of mice were detected using ELISA kits (R&D Systems) following the supplier's protocols and quantified at OD450 value immediately with the microplate reader (Multiskan FC, Thermo Fisher Scientific).

| Masson staining
The lung tissues were immobilized, embedded, and prepared into 5 µm slices.Masson staining was then conducted on the slices to observe the deposition of collagen fibers utilizing the Trichrome Stain (Masson) Kit (Sigma) following the supplier's instruction.The staining results were analyzed under the microscope (DM2500, Leica) and quantified based on the Ashcroft score. 1811 | Cell culture and cell transfection RAW264.7 cells were acquired from the BeNa Culture Collection and maintained in Dulbecco's modified eagle medium with 10% fetal bovine serum (FBS) (Gibco) at 37°C with 5% CO 2 .In the indicated experiments, RAW264.7 cells were induced by 1 µg/mL of LPS for 2 h or treated by 10, 20, 40, 80, 160 μM of fortunellin for 24 h.

| Cell viability
Cell viability of RAW264.7 cells was measured using the cholecystokinin-octapeptide (CCK-8) method.Cells were seeded into 96-well plates (10 4 cells/well) for 24 h cultivation.Then, 10 µL of CCK-8 was added to cells for 4 h treatment.Absorbance at 450 nm was recorded utilizing the microplate reader (Thermo Fisher Scientific).

| Statistical analysis
Data were shown as mean ± standard deviation (SD), and analysis was completed using SPSS Statistics.One-way analysis of variance (ANOVA) with Fisher's protected least significant difference post hoc test was applied for multiple comparisons.p < .05 was identified as statistically significant.

| Fortunellin attenuates LPS-stimulated ALI
To clarify the function of fortunellin on ALI, the ALI mouse model was first established via induction by LPS.The ALI mice model was then administrated with fortunellin.The structure of fortunellin was presented in Figure 1A.HE staining showed apparent lung tissue damage in ALI mice, while 20 and 30 mg/kg of fortunellin significantly attenuated the lung tissue damage (p < .05, Figure 1B,C).However, 10 mg/kg of fortunellin did not affect lung tissue damage.Besides, the W/D weight ratio of tissues and MPO activity in lung tissue were increased in ALI mice (p < .01),but decreased by fortunellin administration (p < .05, Figure 2A,B).Furthermore, the protein content in BALF and the neutrophils and leukocytes numbers were greatly unregulated in ALI mice (p < .01),but fortunellin treatment reversed this phenomenon (p < .05, Figure 2C-E).Meanwhile, the number of monocytes in BALF was slightly decreased in ALI mice (p < .05),while fortunellin did not affect the monocyte number (Figure 2F).Moreover, fortunellin treatment restored the decreased minute ventilation, airway resistance, and lung volume induced by LPS stimulation (p < .05, Figure 2G).Consequently, fortunellin attenuated LPS-induced lung tissue impairment in mice.

| Fortunellin attenuates LPS-stimulated lung tissue apoptosis
To further study the influence of fortunellin on ALI, lung tissue apoptosis in the ALI model was determined after fortunellin treatment.LPS stimulation promoted cell apoptosis in lung tissue (p < .001, Figure 3A,B).However, fortunellin treatment inhibited the proportion of cell apoptosis in lung tissues (p < .05, Figure 3A,B).Furthermore, the expressions of cleaved caspase-3 and Bax were enhanced, but the Bcl-2 level was downregulated after LPS stimulation (p < .01),which was abrogated after fortunellin administration (p < .05, Figure 3C).Therefore, fortunellin attenuated LPS-induced lung tissue apoptosis in mice.

| Fortunellin attenuates LPSstimulated inflammatory response and collagen deposition in lung tissue
To better elaborate on the action of fortunellin on ALI, the inflammation and collagen deposition in ALI mice were determined after fortunellin treatment.The secretions of IL-1β, IL-6, and TNF-α in BALF were upregulated after LPS stimulation (p < .001),while fortunellin abrogated this phenomenon (p < .05, Figure 4A).Besides, lung tissue collagen deposition was occurred in ALI mice, and fortunellin administration attenuated the collagen deposition (Figure 4B).The Ashcroft score was consistent with Masson staining results (Figure 4C).The levels of α-SMA and collagen I were also enhanced in lung tissues of ALI mice (p < .01)but reduced after fortunellin administration (p < .05, Figure 4D).Thus, fortunellin attenuated LPS-induced inflammation and collagen deposition of lung tissue.

| Fortunellin attenuates LPSstimulated ALI via restraining the TLR4/NF-κB/NLRP3 pathway
To confirm whether fortunellin regulated ALI through modulating the TLR4/NF-κB/NLRP3 pathway, the lung tissue changes of ALI mice were determined after being treated by fortunellin and overexpressed TLR4.Results found that the protection of fortunellin on lung tissue damage in ALI mice was abolished by overexpressed TLR4 (p < .05, Figure 6A).Besides, the inhibitory influences of fortunellin on the cleaved caspase-3 in lung tissue and the secretions of IL-β, IL-6, and TNF-α in BALF of ALI mice were also reversed by overexpressed TLR4 (p < .05, Figure 6B-D).Furthermore, overexpressed TLR4 abrogated the alleviative function of fortunellin on the levels of α-SMA and collagen I in lung tissues of the ALI model (p < .05, Figure 6E).Collectively, fortunellin attenuated LPS-stimulated ALI by blocking the TLR4/NF-κB/NLRP3 pathway.

| DISCUSSION
ALI is the prevalent respiratory disease of acute inflammation with high morbidity and mortality. 1,2An effective strategy for ALI therapy still needs to be developed due to the continuously increasing drug resistance and the emergence of new pathogens. 6ortunellin has anti-inflammation properties, 7,9,10 while its role in ALI progression remains elusive.Hence, we investigated the function of fortunellin on ALI development and studied the potential mechanism.
LPS is the principal part of the outer membranes of gram-negative bacteria, and it triggers lung impairment and inflammation. 19The LPS-stimulated ALI model is accepted for studying ALI because it simulates the pathological events in ALI progression, including inflammation and histological changes. 19,20Therefore, the LPSinduced ALI model was selected to perform follow-up experiments in this study.In this model, the lung tissue structure was severely damaged, reflected by destroyed pulmonary architecture, inflammatory cell infiltration, and intra-alveolar edema, consistent with the previous study. 19Besides, the W/D weight ratio of lung tissues, MPO activity in lung tissue, the protein content in BALF, and the neutrophils and leukocytes numbers in BALF were increased in ALI mice.The W/D weight ratio can assess the magnitude of edema. 6MPO content reflects neutrophil infiltration. 19These results confirmed lung tissue damage, edema, and inflammatory cell infiltration in ALI mice.These findings indicated that the LPSstimulated ALI mice model was effectively constructed.Furthermore, we found that the LPS-stimulated lung impairment, edema, and inflammatory cell infiltration were alleviated by fortunellin administration.
Apoptosis, a form of programmed cell death, is imperative for the selective clearance of cells. 21Pulmonary cell apoptosis exerts a vital role in ALI pathogenesis. 213][24] Chen et al. found that cell apoptosis was enhanced in the lung tissues of ALI mice. 22Similarly, cell apoptosis of lung tissues was observed in this study.Interestingly, the promoted cell apoptosis triggered by LPS was suppressed after fortunellin treatment.This finding was consistent with the reported studies. 9,10ong et al. revealed that fortunellin repressed epithelial cell apoptosis by controlling phosphatase and tensin homolog deleted on chromosome 10 expression in colitis. 9Agrawal et al. predicted that fortunellin might inhibit the apoptosis pathway and protect against tissue damage in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected tissues. 10Thus, these findings revealed that fortunellin attenuated LPSinduced lung tissue apoptosis.
The excessive inflammatory response is a primary feature of ALI. 25 In LPS-stimulated ALI, TLR4 identifies LPS and subsequently facilitates the NF-κB activation. 21F-κB activation triggers the secretions of inflammatory cytokines, such as TNF-α, IL-6, and IL-1β.21 In the present research, the elevated secretions of TNF-α, IL-6, and IL-1β in ALI mice were reduced by fortunellin.In other words, fortunellin suppressed the inflammation stimulated by LPS.Similarly, Zhao et al. found that the proinflammatory cytokines were dramatically reduced by fortunellin in high fructose-induced diabetic heart injury.7 Xiong et al. demonstrated that fortunellin inhibited excessive inflammation in colitis. 9 ollagen deposition was the feature of fibroproliferation which is a part of the normal repair response process, and patients may develop fibrosis if this process is ineffective or persists unabated.26,27 Recent evidence revealed that excessive lung collagen deposition in lung tissue usually occurred after ALI, even at the onset of ALI, and contributed to lung fibrosis.28,29 In this study, lung tissue collagen deposition was also observed, which is consistent with previous studies.30,31 Besides, fortunellin reduced lung tissue collagen deposition.Together, these findings revealed that fortunellin attenuated LPSinduced inflammatory response and collagen deposition of lung tissue.
In conclusion, fortunellin attenuates LPS-induced lung impairment, apoptosis, inflammation, and collagen deposition by restraining the TLR4/NF-κB/NLRP3 pathway.This research reported the function of fortunellin on LPS-stimulated ALI pathogenesis and elaborated on the mechanism.Fortunellin may be a valuable drug for ALI therapy.The limitation of this study is that we did not investigate the effect of fortunellin on oxidative stress and autophagy in ALI, which will be explored in the following study.

F I G U R E 1
Fortunellin attenuates lipopolysaccharide (LPS)-induced lung tissue damage.Mice were divided into sham, LPS, LPS + 10 mg/kg FOR, LPS + 20 mg/kg FOR, and LPS + 30 mg/kg FOR groups.The mice in the LPS group were treated by LPS for 12 h.The mice in LPS + FOR-treated groups were treated with the indicated dose of fortunellin before 1 h of LPS administration.(A) The structure of fortunellin was presented.(B) The lung injury score was determined in acute lung injury (ALI) mice after treatment with 10, 20, and 30 mg/kg of fortunellin.(C) hematoxylin-eosin (HE) staining evaluated the lung tissue damage in ALI mice after treatment with 10, 20, and 30 mg/kg of fortunellin.**: p < .01versus sham group; # : p < .05versus LPS group; ## : p < .01versus LPS group.

F I G U R E 2
Fortunellin attenuates lipopolysaccharide (LPS)-induced acute lung injury (ALI).The wet/dry (W/D) weight ratio of lung tissues (A), the content of myeloperoxidase (MPO) in lung tissue (B), the protein content in bronchoalveolar lavage (BALF) (C), the number of neutrophils in BALF (D), the number of leukocytes in BALF (E), the number of monocytes in BALF (F) and the minute ventilation, airway resistance, and lung volume (G) were determined in ALI mice after treated by fortunellin.**: p < .01versus sham group; ***: p < .001versus sham group; # : p < .05versus LPS group; ## : p < .01versus LPS group.